Use of probiotic lactic acid bacteria for balancing the skin&#39;s immune system

ABSTRACT

The present invention pertains to the use of probiotics for the preparation of a carrier for balancing the skin&#39;s immune function. In particular, the present invention pertains to the use of probiotic micro-organisms for balancing the skin&#39;s immune function under stress conditions, such as a exposure to ultraviolet radiation, specifically for enhancing the skin&#39;s immune activity and reducing the tendency to develop allergic reactions under such conditions.

The present invention pertains to the use of probiotics for thepreparation of a carrier for balancing the skin's immune function. Inparticular, the present invention pertains to the use of probioticmicro-organisms for improving the skin's immune function under stressconditions, leading to immune suppression, specifically for normalizingthe skin's immune activity and reducing the tendency to develophyper-reactions under such conditions.

The continuous decrease of the atmosphere's ozone layer with theconcurrent increase of ultraviolet radiation reaching the planet'ssurface has attracted a great deal of interest in its potentialconsequence on human health. Although exposure to ultraviolet radiationis needed for humans to produce vitamin D, growing evidence suggeststhat extensive exposure to sun-light, in particular to ultravioletradiation, causes a variety of problems in the skin, including inductionof certain skin cancers and induction of accelerated skin ageing(photoageing).

It is presently hypothesized that the primary factor of generating skincancer is a mutational damage in the DNA of the generative cells in theskin caused by ultraviolet radiation while UV-light induced injury tothe skin's immune system obviously seems to supply a second factornecessary for the further development thereof. In a healthy system earlymalignant cells are eliminated by the normal functioning of the skin'simmune system. Yet, upon ultraviolet radiation the skin's immune systemseems to undergo suppression and cannot perform its usual surveillancefunction any more. As a consequence very early skin cancer cells are noteliminated, which situation will eventually lead to the malignant cellsto escape the immune system and develop to tumours.

In addition to these established health concerns, research has alsoprovided evidence suggesting that exposure to ultraviolet radiation maynegatively affect a variety of immune responses in living beings bothlocally, within the UV-irradiated skin, and also systemically, i.e. atsites distant from the irradiated skin Exposure of mice to UV-Bradiation has been found to interfere with the rejection of UV-inducedskin cancers and the induction of delayed type and contacthypersensitivity (DTH, CHS) responses initiated at unirradiated sites.These forms of immuno-suppression found upon ultraviolet radiation areconsidered to be associated with the induction of antigen-specificsuppressor T-lymphocytes. The DTH response is particularly importantbecause this T-lymphocyte-mediated immune reaction is responsible forprotection against many chronic infectious diseases.

Current experimental evidence implicates soluble substances derived fromUV-irradiated keratinocytes as probable mediators of UV-induced systemicsuppression of DTH- and CHS-responses. Based on an in vivo actionspectrum for systemic suppression of CHS in mice, it has been proposedthat urocanic acid, a deamination product of histidine, present in thestratum corneum, is one of the photoreceptor for this form of UV-inducedimmuno-suppression.

Apart from suppressing the skin's immune system ultraviolet radiationhas been found to also induce inflammatory and irritant effects in theskin, which may eventually result in the development of erythema, edemaand/or flaking or scaling (hyperkeratosis) of the skin. Theseinflammatory and/or irritant reactions are quite separate anddissociable from the first mentioned one, in that in contrast to thesuppression of the immune system these reactions are rather related to astimulation thereof.

In the art there have been several attempts to alleviate the detrimentaleffects of ultraviolet radiation on the skin, such as by usingsunscreens or other particular pharmacological agents.

In J. Invest. Dermatol., 97 (1991), 624-628 it is reported that topicalapplication of ultraviolet radiation-absorbing compounds (sunscreens) iseffective in preventing ultraviolet radiation-induced erythema and edemabut cannot prevent UV-light induced immuno-suppression. This finding wasconfirmed by several other studies, according to which sunscreens seemto prevent inflammation and/or irritation but do not provide completeprophylactic protection against the immuno-suppressive effects ofultraviolet radiation.

Furthermore, known pharmacological agents which are commonly employedfor the treatment of irritated and inflamed skin, such ascorticosteroids, indomethiacin or acetylsalicylic acid were found to bewithout effect in treating the UV-light induced suppressed condition ofthe skin's immune system when they are applied after the injury ismanifest. As proposed by Bergstrasser et al. in Immunology 46 (1989),219-245, local application of corticosteroids reduce the skin's immuneresponse in general. Although indomethacin has been demonstrated byReeve et al., in Cancer Letters 95 (1995), 213-219 to inhibitphoto-carcinogenesis this effect appears to involve both the initiationperiod and the promotion period of tumour development and thus isthought to be a function of a generalized anti-carcinogenesis effectrather than an effect on the skin immune system. Thus, there appears tobe a pattern whereby agents capable of suppressing inflammation andirritancy may not protect the skin's immune system.

On the other hand agents that proved to be effective against ultravioletradiation induced immuno-suppression did not show any effect inimproving inflammatory and irritant effects caused by the skin'sexposure to ultraviolet radiation. In U.S. Pat. No. 5,302,389 such anagent and a method for the treatment or prevention of UV-inducedimmuno-suppression through the application of liposome-encapsulated DNArepair enzymes is proposed. This agent is, however, not effective fortreating inflammatory and irritant responses of the skin.

Consequently, there is a need in the art for an agent that may reducethe skin's tendency to develop hyper-reactions when being subjected tostress conditions, and which is capable to reduce the effect ofultraviolet radiation on suppressing the skin's immune system.

In the past various publications described micro-organisms exerting abeneficial influence on the individual's well being. According to R.Fuller in the Journal of Applied Bacteriology 66 (1989), 365-378, thesemicro-organisms were designated “probiotics” and were defined as livemicrobes beneficially affecting the host by improving its intestinalmicrobial balance.

Probiotics are non-pathogenic and non-toxigenic organisms, that survivepassage through the stomach and small intestine. Upon continuousingestion by the host they eventually may colonize the gut to asubstantial extent thus competing with other potentially pathogenicbacteria for nutrients and/or attachment sites on the gastro-intestinalwall and reducing their numbers and reducing or preventing infections.

Until now a number of different probiotic micro-organisms have beenfound, which all are reported to exert their effect in the gut via theproduction of toxins, metabolic by-products, short chain fatty acids andthe like.

It has also been demonstrated that the microbiota of thegastrointestinal tract may affect the mucosal immunity within the host.According to Schiffrin et al., in Am. J. Clin. Nutr. 66 (1997), 520, theintestinal epithelial cells, blood leukocytes, B- and T-lymphocytes, andalso accessory cells of the immune system have all been implicated inthe aforementioned immunity to a certain extent. Accordingly, probioticorganisms are considered to interact with the immune system at manylevels including cytokine production, mononuclear cell proliferation,macrophage phagocytosis and killing, immunity to bacterial and protozoanpathogens, and the hie.

Since the biological activity of the probiotics is mainly taking placein/on the gut's mucosa or the adjacent tissue of the individual, theeffect of such micro-organisms was considered to reside mainly in thispart of the body. In this respect, literature provided ample evidencethat ingestion of probiotics by an individual may show some effect inthe gastro-intestial tract. WO 00/41707 discloses a Lactobacillussalivarius strain useful in the prophylaxis or treatment of undesirablegastrointestinal inflammatory activity such as inflammatory boweldisease or irritable bowel syndrome. Further, in WO 00/35465compositions for the oral administration of Lactobacillus and/or otherprobiotic organisms are disclosed, for establishment and maintenance ofa healthy urogenital flora.

During the extensive studies leading to the present invention it was nowsurprisingly found that probiotics do also exert an effect in anindividual's body at a location distant from the region in which theycolonize it. In particular, it has been found that probioticmicro-organisms do also exert an activity on the immune system in theskin of the individual. Accordingly it has been found that uponingestion by an individual they may balance a suppression of the skin'simmune system inherent to exposure to stress, such as physical, chemicalor biological stress, while they may also reduce the individual'stendency to develop inflammatory and/or irritant reactions upon exposureto such a stress condition.

Consequently, according to the broadest aspect the present inventionprovides for the use of probiotics or a culture supernatant thereof forthe preparation of a carrier for balancing the skin's immune function.

For the purpose of the present invention the term “balancing the skin'simmune function” shall be interpreted as normalizing the immune functionunder a stress condition, that leads to a suppression of the immunesystem, that is, maintaining an immune function or immune condition at alevel normally prevailing in the skin even when being exposed to suchstress conditions. A stress condition shall be interpreted to comprisephysical stress, e.g. ultraviolet irradiation of the skin, chemicalstress, such as exposure to chemical agents, or allergenic material, orbiological stress, such as being exposed to or infected bymicro-organisms, such as pathogenic bacteria, viruses, fungi etc. Undersuch conditions the immune system is on one hand suppressed, e.g. whenbeing exposed to irradiation with UV-light, and on the other handover-stimulated, such as when being exposed to irritants and allergens.Further, in the context of this invention culture supernatant thereofshall designate the supernatant of a culture of a probiotic as such orin concentrated form or the active metabolite(s) isolated therefrom.

It has now been found that in case of administering probiotics to anindividual, which may colonize the individual's gut, or a culturesupernatant thereof, the immuno-suppressive effect of certain stressconditions, such as e.g. ultraviolet radiation, is found to be lesspronounced or substantially reduced, while inflammatory and/or irritantreactions do occur only to a diminish degree or not at all.

In the figures,

FIG. 1 shows the inflammatory reaction associated with a contacthypersensitivity reaction in the various mice models examined asdetermined by ear swelling;

FIG. 2 shows the results obtained with an antibody against ICAM-1 in thevarious animal models indicating

FIG. 3 shows the level of TGF-β in the various animal models tested;

FIG. 4 shows the epidermal IL-10 level in the various animal modelstested.

FIG. 5 shows the effects of probiotics on an UV irradiation inducedsuppression of the immune system shown according to a contacthypersensitivity reaction as determined by ear swelling;

FIG. 6 shows the effects of probiotics on an UV irradiation inducedsuppression of the immune system shown as % CHS inhibition.

The present invention provides for the first time that the gut floracomposition may also have a beneficial effect on certain protectivefunctions in regions of the bodies distant from the location at whichthe probiotic micro-organisms colonize the individual, namely the skin.Further, it has been shown that the probiotics may control in the skindivergent processes such as up-regulating the immune system during animmuno-suppressive condition and at the same time down-regulatinghyper-reactions, such as inflammatory or allergic reaction, such aseczema or atopic dermatitis. This holds especially true for elderlyindividuals, who normally have a reduced immunocapacity.

According to a preferred embodiment the probiotics to be included intothe carrier are selected from the group consisting of Lactic acidbacteria, in particular Lactobacilli and/or Bifidobacteria and are morepreferably Lactobacillus johnsonii, Lactobacilus reuteri, Lactobacillusrhamnosus, Lactobacillus paracasei, Lactobacillus casei orBifidobacterium bifidum, B. breve, B. animalis, B. infantis, B.dolescentis B. pseudocatenulatum. Since due to their distinct oxygenrequirement Lactobacilli and Bifidobacteria colonize the gut atdifferent locations a mixture thereof may be used so as to provide abroad coverage.

According to a most preferred embodiment the strains used areLactobacillus johnsonii (La1) deposited under the Budapest Treaty withthe Institute Pasteur and receiving the deposit no. CNCM I-1225 orLactobacillus paracasei (ST11) deposited with the Institute Pasteuraccording to the Budapest Treaty and receiving the deposit no. CNCMI-2116.

The carrier may be any food or pharmaceutical product, or a cosmeticproduct for oral or topical application, wherein the probioticmicroorganism or a culture supernatant thereof may be included. Examplesfor food or pharmaceutical carriers are milk, yogurt, curd, cheese,fermented milks, milk based fermented products, ice-creams, fermentedcereal based products, milk based powders, infant formulae or pet food,or tablets, liquid bacterial suspensions, dried oral supplement, wetoral supplement, dry tube-feeding or wet tube-feeding. For cosmeticproducts lotions, shampoos, creams, such as moisturising creams,sun-screens, after-sun creams or anti-aging creams, and/or ointments areenvisaged, wherein the micro-organism may be included in a live form,semi-active or in deactivated form, e.g. as a lyophilized powder. Alsoculture supernatants of the micro-organisms may be included in thecosmetic products, optionally in concentrated form.

It will be appreciated that when administered to an individual as amicro-organism the probiotic's balancing activity will be dosedependant. Thus, it is envisaged to include as many as from 10⁵ to 10¹²organisms/g product, which organisms may be alive or dead. When using asupernatant of a probiotic's culture the supernatant may be used as suchor may subjected to one or more purification steps prior to inclusioninto the product, so as to concentrate or isolate the activeingredient(s)/metabolite(s). Methods and techniques for purifyingcompounds and detecting the activity thereof in the fractions obtainedare well known to the skilled person.

Though the present invention applies to all living beings, such ashumans and animals, in particular pets, the probiotics are preferablyused for elderly individuals, that generally have a reducedimmuno-capacity.

During the far-reaching experimentation leading to the present inventiona germ-free animal model was utilized focusing on microflora associatedcharacteristics (MAC) vs. germfree animal characteristics (GAC), whichcharacteristics are deemed to be of considerable value, particularly inallergic/inflammatory diseases with skin manifestations.

The following examples further illustrate the invention without limitingit thereto.

EXAMPLE 1 Balancing Hypersensitivity Reactions

In this experiment the activity of probiotics (dead or alive) on theskin's immune system under a chemical stress situation was evaluated. Inthis respect, animals were sensitized with a chemical compound(dinitrochlorobenzene) and the effect of such a (chemical) stresssituation in axenic animals was compared to the effect in animals, thegut of which contained probiotics only. The level of a hypersensitivityreaction developed against DNCB was evaluated by several parameters (seebelow).

For the experiments male mice C3H (LPS+), germ free and supplied by theResearch Center Orleans (CNRS Orleans, France) were assembled in fourgroups of eight mice each. The groups reflected different statuses ofthe gut and were designated “C” (group with a Conventional (normal)microflora), “LV” (group with a microflora consisting of LiVing Lacticacid bacteria only), “LD” (group fed with dead probiotics (LactobacillusDead) and “A” (germ free (Axenic) mice); all groups were maintained inisolated cages with group C being maintained under normal conditions.

The probiotic administered to the mice was the lactic acid bacteriumST11 (obtainable from the Institute Pasteur under the accession no. CNCMI-2116) which was given in a live form in a solution of 10⁸ cfu/ml(cfu=colony forming unit), or used dead or inactivated by irradiation ina solution of 2.1 g/25 ml in water when forced feeding was applied or inwater at 3.3 g/l (below).

To obtain mice having a microflora essentially consisting of live lacticacid bacteria only, the germ free animals (supra) were force-fed twiceon day 7 and day 8 with 0.5 ml of a solution containing 10⁸ cfu/ml each(group LV). Alternatively, in order to obtain mice having a gutmicroflora with the probiotic strain, inactivated by irradiation, thegerm free animals were force-fed from day 36 to day 38, from day 41 today 45, from day 48 to day 49 and from day 52 with 0.2 ml of a solutioncontaining 2.1 g/25 ml inactivated microorganisms each day indicated.Starting from day 55 the above solution was replaced by a solutioncontaining 3.3 g/l inactivated micro-organisms, which solution wasprovided until the end of experiment in drinking water (group LD). Toobtain a conventional (normal) gut-microflora, the germ free animalswere transferred three days after arrival into a common animal cage withnormal feeding so as to develop a normal microflora. All the animals ingroups A, LV and LD were examined on a regular basis for their fecalmicrobial composition, to ensure the maintenance of their specific gutstatus.

The animals in the different groups were sensibilized by topicallyapplying on each flank 50 μl of 1% dinitrochlorobenzene (DNCB) on day 45and 50, respectively. Five days after the last sensitization the animalswere challenged on three consecutive days (on day 55, day 56 and day 57)by applying on the right ear 25 μl of 1% DNCB and on the left ear 25 μlof the carrier (olive oil) for all mice groups.

On day 58 the effect of the hypersensitivity reaction brought about bythe above treatment with DNCB was examined. In this respect, thethickness of the right and left ears of each of the animals was measuredon day 58 and the difference in the thickness between the two ears ofthe same animal has been calculated. The results are shown in FIG. 1 andare summarized in table I below:

TABLE 1 Group Δ ear thickness 10⁻² mm ± s/√n A 22.9 ± 2.2 LD 19.0 ± 3.6LV 17.0 ± 3.3 C 12.1 ± 2.8

From the above it becomes evident that a hypersensitivity reaction asmeasured according to the ear thickness increases in the order

C<LV<LD<A

From these results, it can be concluded that a conventional intestinalflora permits a regulation of the inflammation as compared to micelacking such a microflora. In addition, it may be seen that theimplementation of the probiotic micro-organism ST11 alone causes areduction of the inflammation caused by DNCB, i.e. a tendency to reducethe edema of the ears of the animal exclusively with a lactic gut flora.This finding is surprising, since the gut normally consists of anassembly of different micro-organisms, that all fulfil different tasks.

Moreover, in order to obtain biochemical data on immunological relevantresponses on the molecular level to the stress situation cryo-sectionsof the right ear were prepared and examined for the level of ICAM-1 andTGF-β. ICAM-1 is a pro-inflammatory marker while TGF-β is ananti-inflammatory marker. Consequently, it is expected that the stresscondition induced will result in the ICAM-1 level being high and theTGF-β level being low.

For the experiments the rat anti-mouse ICAM-1 antibody (clone KAT-1) at1/25 (Caltag Laboratories, CA, USA) was used, then rabbit ant-rat FITCat 1/700 (Dako, Ca, USA). For the determination of TGF-β rabbitanti-human/mouse TGF-β (V) at 1/20 (Santa Cruz Biotechnology, Ca, USA),was utilized, then swine anti-rabbit-FITC at 1/1000 (Dako, Ca, USA).

As may be seen from FIG. 2, in axenic animals a high level of theinflammatory cytokine ICAM-1 could be detected, while in animals treatedwith the living probiotic a decreased level of ICAM-1 could be observed,comparable to that found in animals having a conventional, i.e. normalmicroflora. Also animals treated with inactivated probiotic showed adecreased level of ICAM-1. According to this finding the following ordermay be set up

C=LV>LD>A

Again, based on the finding relating to this inflammatory marker animalshaving a gut microflora consisting of the lactic acid bacterium aloneshowed the same low level of ICAM-1 as did mice having a conventionalmicroflora.

In contrast thereto, FIG. 3 shows that the level of theanti-inflammatory marker TGF-β was decreased in axenic animals ascompared to animals having a conventional microflora. Yet, animalshaving a microflora with the living clearly showed an increased level ofTGF-β as compared to axenic animals. The order to be set up for thismarker is

C>LV>LD>A

In addition, skin samples were taken from the back of the mice andsubjected to an extraction procedure according to the method describedin Peguet et al., Br. J. Dermatol. 133 (1995), 661, which document isincorporated herein by way of reference. After taking skin samples, theywere transferred on ice into an extraction buffer 10 mM Tris HCl, pH7.4; 2 mM MgCl; 150 mM NaCl; 1% Triton X100; 2 mM PMSF) and subjected toan ultrasonication treatment. Also serum samples were taken from theanimals. The serum samples and skin extracts were examined for theirIL-10 level.

For performing the experiments the ultrasensitive ELISA test Cytoscreen®from Biosource International, CA, USA was used. The amount of IL-10 wascalculated according to a standard curve (0.625 to 80 pg/ml)

For the extracts the results are reported according to the amount ofproteins (Lowry method) contained in the extracts based on the totalamount of cellular material. The results are expressed in pg IL-10 andshown in FIG. 4 and table II below.

TABLE II Groups IL-10 pg/mg A  6.3 + 2.2 LD  6.8 + 1.7 LV 11.1 + 1.8 C21.2 + 3.2

As may be clearly seen, the amount of IL-10 is the highest in micehaving a conventional microflora with the group treated with liveprobiotic being superior to axenic mice. The following order may be setup:

C>LV>LD>A

Based on the finding in this experiment it may clearly be seen that thedifferent parameters tested (TGF-β, ICAM-1, IL-10) confirm the resultsfound with the difference in ear thickness, i.e. a regulation ofinflammatory reactions by implanting a lactobacillus microflora.

EXAMPLE 2

In this experiment the UV-light induced suppression of the immune systemin the skin, as perceptible by an diminished development of ahypersensitivity reaction, was used to investigate the potential ofprobiotics to essentially restore the immune system's capability torespond to stress situations, such as being exposed to an allergenicsubstance, in a normal way.

Three test groups of animals were set up. The first group of animals wastreated with a compound known to elicit hypersensitive reactions,dinitrofluorobenzene (DNFB), and the reaction of the skin's immunesystem thereto was determined. The second group was subjected to UVirradiation prior to being exposed to the compound above and the effectof said exposure on the immune reaction under these conditions wasassessed. The third group received probiotics in living or inactivatedform, or a culture supernatant thereof, and was exposed to UV-light. Inthis group the effect of the probiotics on the restoration of the immuneresponse was evaluated. For performing the experiment female Skh1/hrmice (obtained from Charles River Laboratories (France)) aged between 8and 10 weeks were used. As probiotics administered to the differentgroups the products listed in table III were utilized:

TABLE III Product 1 culture medium Product 2 live La1 (obtainable fromInstitute Pasteur, CNCM I-1225) Product 3 inactivated La1 Product 4supernatant of La1 Product 5 live ST11 (obtainable from InstitutePasteur, CNCM I-2116) Product 6 inactivated ST11 Product 7 culturesupernatant of ST11

The probiotic samples were provided in frozen aliquots of 1.5 mlcontaining 10⁹ cfu/ml. As a control the culture medium (product 1) wasutilized.

32 groups of 10 mice each were assembled. Animals were fed with therespective products starting 10 days before day 0, the day of exposingthe animals to UV-light, and continuing such feeding until day 12, onwhich the challenge with DNFB took place. Consequently, the productswere fed to the animals for 23 days in total. The products 1 to 7(supra) were force-fed to groups 5 to 32 at a dosage of 100 μl/animalcorresponding to about 10⁸ cfu/animal/day. Table IV lists the productsand the groups receiving them.

TABLE IV Product 1 (culture medium) groups 5-8 Product 2 (live La1)groups 9-12 Product 3 (inactivated La1) groups 13-16 Product 4(supernatant of La1) groups 17-20 Product 5 (live ST11) groups 21-24Product 6 (inactivated ST11) groups 25-28 Product 7 (supernatant ofST11) groups 29-32

On day 0 the mice were lightly anestesized with isofluorane/oxygen andsubsequently irradiated by means of a sun-simulator containing a Xenon1000 W (Oriel) lamp including a dichroic mirror (Oriel, Stafford, USA)equipped with a WG 320/1 mm thick filter and a UG11 filter/1 mm thick(Schott). This filtered source provided a simulated solar US spectrum(290-400 nm) that almost eliminated all visible and infrared radiation.The amount of radiation as determined by means of radiometer ARCC 1600(Osram) was 1.95 mW/cm² at UVB and 9.35 mW/cm² at UVA. The spectrumapplied conforms to the norm of according to SPF COLIPA.

Mice of the groups 3, 4, 7, 8, 9, 11, 12, 15, 16, 19, 20, 23, 24, 27,28, 31, 32 were irradiated, with the ears being protected (day 0). Theyreceived a singular dose of 2.5 MED.

On day 5 and day 6 a hypersensitivity reaction was induced by topicallyapplying on the abdomen 50 μl acetone (groups 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31) and DNFB (0.3% in acetone; groups 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32), respectively.

On day 12, 5 μl of a 0.2% DNFB solution in acetone was applied on theright ear of all mice. The animals were weighed at the beginning (day 0)and at the end of the tests (day 13).

The evaluation of the inflammatory reaction was performed 24 hours afterexposure to UV-light, i.e. on day 1. Two parameters were assessed,namely the intensity of the erythema on the back by means of clinicallydetermining the erythema and the edema, with calculating the means ofeach group and the increase of the thickness of the skin at the back,with calculating the means of the non-irradiated and the irradiatedanimals.

The determination of the skin thickness at the right and the left earwas performed on day 13. The difference of the skin thickness of theears of the same animal and the medium value of each group wascalculated. The results shown in the table V below are graphicallydepicted in the FIGS. 5 and 6.

TABLE V Hypersensibility reaction as determined by the ear thickness Δear thickness % Inhibition Irradiation épaisseur of HSC MED TreatmentInduction 10⁻² mm ± s/{square root over (n)} by irradiation 1 0 —Acetone 1.1 ± 0.2 2 0 — DNFB 0.3% 19.3 ± 0.4  3 2.5 — Acetone 1.0 ± 0.24 2.5 — DNFB 0.3% 7.5 ± 0.2 61.1 5 0 Culture medium Acetone 1.0 ± 0.1 60 Culture medium DNFB 0.3% 19.0 ± 0.2  0 7 2.5 Culture medium Acetone1.0 ± 0.2 8 2.5 Culture medium DNFB 0.3% 7.4 ± 0.1 61.6 9 0 La1 aliveAcetone 1.1 ± 0.3 10 0 La1 alive DNFB 0.3% 18.9 ± 0.2  0 11 2.5 La1alive Acetone 1.3 ± 0.2 12 2.5 La1 alive DNFB 0.3% 15.4 ± 0.2  20.2 13 0La1 dead Acetone 1.3 ± 0.2 14 0 La1 dead DNFB 0.3% 19.4 ± 0.3  0 15 2.5La1 dead Acetone 1.4 ± 0.1 16 2.5 La1 dead DNFB 0.3% 9.6 ± 0.3 50.3 17 0Culture supernatant of Acetone 1.1 ± 0.2 La1 18 0 Culture supernatant ofDNFB 0.3% 19.5 ± 0.4  0 La1 19 2.5 Culture supernatant of Acetone 1.1 ±0.3 La1 20 2.5 Culture supernatant of DNFB 0.3% 7.4 ± 0.1 61.7 La1 21 0ST11 alive Acetone 0.6 ± 0.2 22 0 ST11 alive DNFB 0.3% 18.8 ± 0.2  0 232.5 ST11 alive Acetone 1.1 ± 0.2 24 2.5 ST11 alive DNFB 0.3% 13.2 ± 0.3 31.6 25 0 ST11 dead Acetone 1.4 ± 0.2 26 0 ST11 dead DNFB 0.3% 19.4 ±0.4  0 27 2.5 ST11 dead Acetone 1.1 ± 0.3 28 2.5 ST11 dead DNFB 0.3%10.6 ± 0.3  45.1 29 0 Culture supernatant of Acetone 1.0 ± 0.2 ST11 30 0Culture supernatant of DNFB 0.3% 19.3 ± 0.4  0 ST11 31 2.5 Culturesupernatant of Acetone 1.2 ± 0.2 ST11 32 2.5 Culture supernatant of DNFB0.3% 11.0 ± 0.2  43 ST11

As expected, exposure to UV-light systemically suppressed ahypersensitivity reaction to DNFB in mice receiving no treatment at alland in control mice, receiving the culture medium alone. Those micereceiving live probiotic micro-organisms or culture supernatants thereof(conditioned media) clearly showed a recovery of the immune system andexhibited a hypersensitivity reaction against DNFB. From these findingsit becomes obvious that probiotics and to some extent also theirmetabolites, present in the culture supernatant, are clearly capable tomodulate the immune system in an UV-light induced suppressed conditionsuch that the immune response is essentially restored.

1-10. (canceled) 11: A method for preparing a treatment product forbalancing a skin's immune function of an individual, the methodcomprising: adding an effective amount of a probiotic lactic acidbacteria or a culture supernatant thereof to an ingestable carrier toproduce the treatment product, the effective amount of the probioticlactic acid bacteria or a culture supernatant thereof capable ofdown-regulating inflammatory or allergic reaction in the skin induced byultra-violet irradiation of the skin and up-regulating the immune systemin the skin during an immuno-suppressive condition induced byultra-violet irradiation of the skin. 12: The method of claim 11,wherein the lactic acid bacteria is selected from the group consistingof Lactobacilli, Bifidobacteria and combinations thereof. 13: The methodof claim 11, wherein the lactic acid bacteria is selected from the groupconsisting of CNCM I-1225, CNCM I-2116 and combinations thereof. 14: Themethod according to claim 11, wherein the ingestable carrier is selectedfrom the group consisting of milk, yogurt, curd, cheese, fermentedmilks, milk-based fermented products, ice-creams, fermented cereal basedproducts, milk based powders, infant formulae, tablets, liquid bacterialsuspensions, dried oral supplement, wet oral supplement, dry tubefeeding, wet tube feeding, pet food, pet lotions, shampoos, creams,ointments and combinations thereof. 15: The method according to claim11, wherein the probiotic lactic acid bacteria is present in the carrierin an amount of from about 10⁵ to about 10¹² cfu/g carrier. 16: Themethod according to claim 11, wherein the individual, the skin immunefunction of which is balanced, is selected from the group consisting ofhuman being and pets.